The main conventional techniques which are related to the present invention will be described below.
Currently, integrated substrates for bioassays, i.e., so-called DNA chips or DNA microarrays, (hereinafter referred to as “DNA chips”), in which selected DNA is microarrayed by microarray technology, have been used for the analysis of gene mutations, SNP (single nucleotide polymorphism) analysis, gene expression frequency analysis, etc., and have also started to be used extensively for the development of new drugs, clinical diagnosis, pharmacogenomics, forensic medicine, and other fields.
The DNA chips are characterized in that it is possible to extensively analyze intermolecular interactions, such as hybridizations, because various types and a large number of DNA oligomer chains, cDNA (complementary DNA), etc., can be integrated on glass substrates or silicon substrates.
One example of analytical methods using DNA chips will be briefly described below. That is, PCR amplification is performed in which mRNA extracted from cells, tissues, etc., is incorporated by reverse transcription-PCR or the like into DNA probes solid-phased on a glass substrate or silicon substrate, and hybridization is performed on the substrate. Fluorescent light measurement is then carried out with a suitable detector.
DNA chips can be classified into two types. In the first type of chip, oligonucleotides are synthesized directly on a given substrate using photolithography by applying semiconductor exposure technology. A typical example is the one manufactured by Affymetrix, Inc., U.S.A. (for example, refer to U.S. Pat. No. 5,445,934). In this type of chip, although the integration degree is high, DNA synthesis on the substrate has limitations, and the length is limited to about several tens of bases.
In the second type of chip, which is also referred to as “the Stanford method”, the chip is fabricated by dispensing and solid-phasing prepared DNA on a substrate using a split taper pin (for example, refer to U.S. Pat. No. 5,807,522). In this type of chip, although the integration degree is lower than that of the former type, it is possible to solid-phase about 1 kb of DNA fragments.
Recently, biosensor technology has been advancing, in which a selected detecting substance is solid-phased on a fine detection surface site provided on a thin plate, which is referred to as a biosensor chip, such as a protein chip, and a microvolume of solution containing a target substance is allowed to flow toward the detecting substance, and then the interaction between the two substances is observed and analyzed based on the surface plasmon resonance principle, quartz crystal oscillator principle, or the like. This technology is becoming useful for analyzing interactions between substances, such as antibody-antigen reactions and hormone responses.
In the conventional DNA chip technology and biosensor technology, however, interactions between substances, such as hybridization reactions and antibody-antigen reactions, are carried out by solid-phasing (immobilizing) detecting nucleotide chains, such as DNA probes, and proteins, etc., on two-dimensional, small detection regions of substrates. Consequently, the interactions are carried out mainly based on the Brownian movement of the reaction products under not necessarily favorable reaction conditions in which freedom of the reaction products is limited spatially and there is also a possibility that steric hindrance may occur during the reactions. Therefore, the conventional DNA chip technology and biosensor technology have technical problems, i.e., low interaction efficiency and long reaction time.
Furthermore, in the known DNA chips, etc., sample solutions are only dripped onto predetermined spot sites (detection regions) on substrates, and no devices are used to relatively align the target substances contained in the sample solutions and the detecting substances immobilized on the spot sites.
Accordingly, it is a principal object of the present invention to provide a bioassay method, a bioassay apparatus, and a bioassay substrate that can be advantageously used in the bioassay method and the bioassay apparatus, in which, by controlling the electric field formation in the reaction region where an interaction between substances, such as a hybridization, is carried out, the detecting substance and the target substance are relatively aligned with each other, and the structures of the substances are adjusted, thereby increasing the efficiency of the interaction.